DS DNA Break repair Flashcards by c k

Between NHEJ and HR what is the real big decision about which pathway it is going to go come from…what?

Protect the ends or res-sect the ends…

HR requires long single stranded 3’ end (resulting from resection) so if the ends are protected we cannot do HR

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Loss of function primarily effects which systems

immune
nervous
reproductive

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endogenous DS break causes (3)

immune system rearrangements (ab)
single strand breaks during DNA replication
meiosis - recombination

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exogenous causes of double strand breaks (2)

ionizing radiation (cosmic rays and soils)
medical imaging and treatment

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DSB occur during VDJ recombination

what is this?

v region of ab recognition site recombines next to D in heavy chain or J in light chain - this leads to a huge increase in the number of ab recognition potential

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DSB during DNA replication

How does this happen?

The replication fork stalls at a single strand break …then…that end becomes a free end and replication fork collapse leads to double strand break (one ended double strand break)
- the whole system has to back up and repair that break before replication can continue

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Are double strand breaks required in meiosis?

Yes, at least one recombination even during prophase1 between homologs must occur for successful meiosis and actually there are multiple recombination events during this time

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Two pathways to repair DNA DSBs

Non homologous end joining (NHEJ)

Homologous Recombination

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Is NHEJ a perfect system?

No… it is often imperfect with the loss of a few nucleotides

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When does NHEJ occur?

Throughout the cell cycle

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Can NHEJ join any ends?

No we want to regulate the fusion

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What imperfect system (DSB repair) does immune system take advantage of?

NHEJ - because we lose DNA in the middles and this is somewhat random - increasing the number of AB sites (non-templated mutations as consequence of end joining)

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Is Homologous Repair perfect?

For testing purposes…YES

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Does homologous repair occur with homolog or sister chromatid

Sister chromatid (that’s why it’s perfect)

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Since homologous repair occurs with the sister chromatid, when is it limited to occuring?

S and G2 of the cell cycle

This is when the sister chromatid exists :)

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Why do we have mechanisms to prevent HR outside of S and G2…

Because if we do HR outside of S and G2 we will be using the homolog (from the other parent) and this can (and i think will) lead to loss of heterozygosity

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When you sequence cDNA from the light chain / or heavy chain of antibody and then compare to the genome, what would you find

You would find that in the genome those pieces are not together

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What is the name of the enzyme that initiates double strand breaks in b and t cells undergoing maturation?

rag recombinase (derived from transposable element)

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Rag recombinase -

what does it’s unpredictable and imprecise repair by NHEJ contribute to?

antibody diverstiy

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NHEJ

What is the initial step?

Recognition of the double strand break by KU

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What is Ku?

Ku is the protein that recognizes and binds DSBs

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What does Ku do?

Ku recruits DNA-PKcs to the double strand break

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So…Ku increases the affinity of DNA-PKcs for the DSB by over 100 fold - thus when Ku recognizes DSB it calls in DNA-PKcs… this in turn calls in what?

Artemis

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What is Artemis

Artemis is the protein called in by the KU-DNA-PKcs complex to fix DSB

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Which protein is responsible for processing the DSB ends?

Artemis

``` So we've called in 1. Ku 2. DNA-PKcs 3. Artemis Now what do we need to call in ? ```

Well Artemis processed the DSB ends Now we need to polymerise the DNA in order to fill in the gaps This will be done by polymerase

``` So we've call in 1. Ku 2. DNA-PKcs 3. Artemis 4. Polymerase Who's left? ```

Now we need ligase to restore the continuous phosphodiester backbone Specifically (LIG-4)

is Artemis and endo or exo nuclease?

BOTH!

What direction exonuclease is artemis?

5'-->3'

What does being an exonuclease enable artemis to do?

It can make blunt ends when there is a 5' overhang

Does artemis require activation

YES AND NO Exonuclease activity does not Endonuclease activity does

What activates artemis endonuclease activity

PKcs

What kind of polymerases are called in after artemis has processed the DSB?

DIRTY :) Lambda and Mu They are known to make lots of mutations when they polymerise their strands

Is NHEJ a linear sequence of events?

NO! | variable nuclease and polymerase activity prior to ligation can result in variable outcomes

So if we have one DSB, is our NHEJ repair always going to be the same?

No... we can cause a DSB at same site and recover different structures following NHEJ due to variability of nuclease, polymerase, followed by ligase activity MICROHETEROGENEITY caused by NHEJ

AB programmed DSB ... which enzyme?

Rag Recombinase

Meiosis programmed DSB... which enzyme?

SPO11

What kind of cells express SPO11?

meiotic

What is SPO11

An ENDOnuclease that causes double strand breaks in meiosis

What kind of overhang does SPO11 leave?

3'OH (on top and bottom) | This will be the substrate for homologous recombination

Quantity of SPO11 in the cell?

Exists in many more copies than there are breaks

What controls SPO11 acivity?

ATM

What kind of enzymes are ATM and PKcs?

PIKKs | phosphoinositol-3-kinase like kinases

If you make a mutation in ATM, what happens to SPO11

You get way more attacks and cleavages by SPO11 than you need (gonadal insufficiency = ataxia telangiectasia mutated)

When do we get DSB by SPO11?

ONLY IN MEIOSIS

Why is HR important in meiosis

necessary for proper segregation of homologous chromosomes | also important for genetic diversity

Is it ever important to have HR between homologous chromosomes

YES.. ONLY IN MEIOSIS

How does DSB repair by HR work when there is a single-strand nick during DNA replication?

(THERE IS A PAUSE WHERE THE STRAND INVASION DISPLACED D LOOP BECOMES NEW TEMPLATE FOR CONTINUING THE DNA SYNTHESIS) (ONCE IT GETS PAST THE BREAK, IT CAN RESOLVE BACK AND CONTINUE AS BEFORE) 1. exonuclease degrade 5' end to leave overhanging 3' 2. overhanging 3' can invade (3'-->5') sister chromatid and use a template for a bit 3. this breaks the sister and template 4. additional synthesis 5. replication fork can restart

When we have DSB occur and get a complete break across both strands how do we fix it?

1. Degrade the 5' end of both strands 2. Resulting 3' overhang can invade the D-loop of the sister chromatid (stretching from 3'-->5') 3. 3'OH can extend in from 5'-->3' using sister chromatid as template until break point is reached 4. ligation

Key proteins regulating DSB repair via HR

BRCA1 - required for proper HR selection - recruits complex that does endonuclease cleavage and enables exonuclease to get rid of 5' end RAD51 - recruits BRCA2 - which does strand invasion and D-loop

Alternative outcomes for repair by HR | GERM CELLS VS. SOMATIC

GERM bias to use homolog and resolve via DSBR -->assure recombination SOMATIC bias to use sister chromatid and resolve via SDSA --> avoid recombination and LOH

When does the Holliday junction resolution become relevant?

When we would be using the homologous chromosome rather than than the sister

What are the two possible resolutions of the Holliday junction?

Recombined | Gene conversion

When the homologous chromosome, not the sister chromatid, is involved --> what is possible?

Loss of heterzygosity (LOH) and I think inevitable, (At least where you used it as a template)

What is SDSA?

Synthesis dependent strand annealing

What do we avoid with SDSA?

Recombination and LOH (non-crossovers)

Gene conversion

The process by which one DNA sequence replaces a homologous sequence such that the sequence becomes identical after the conversion event

http://www.web-books.com/MoBio/Free/Ch8D4.htm | Illustrates conversion vs crossover

good explanation as she was confused

What sense DSB in the case of NHEJ

What is responsible for signal transduction in DSB (controls Spo11) in meiosis

ATM

Significance of Wortmannin

inhibits protein kinases that are important for initiating repair - when we have wortmannin we don't get the proteins needed for DSB repair (i.e. RAD51 / BRCA1)

Who brings in players for DSB repair via HR once activated?

BRCA1

All DSB that occur during DNA replication are fixed via which mechanism

HR - which makes sense because sister is right there

Which mechanism of DSB repair is used for exogenous DNA damage?

Both HR and NHEJ | Depends on timing in the cell cycle

Which mechanism of DSB repair is used for VDJ

NHEJ (Rag recombinase causes)

Which mechanism of DSB repair is used for meiosis

HR (Spo11 causes)

Protein that is poised to push the cell towards NHEJ?

53BP1

Push and pull relationship between which two proteins regulate NHEJ vs HR?

53BP1 (NHEJ) and BRCA1 (HR)

Does BRCA1 depend on activation?

Yes it must be activated by a CDK (cell cycle signaler)

Since BRCA1 depends on activation by a CDK... what is the significance?

Can only be activated at certain points (S/G2) in the cell cycle

When CDK is not present (i.e. G1) and BRCA1 cannot be activated, what mechanism do we use?

NHEJ via 53BP1

There are really 4 key regulatory steps in DSB repair... what are they?

1. Signals 2. Sensors 3. Transducers 4. Effectors

Exampes of DSB sensors

PIKKs - ATM - ATR These are protein kinases used to signal DNA damage

2 forms of genetic exchange via Holliday junction resolution

Gene conversion | Cross over